purifying dna from gel slices

vilimf01 at mcrcr6.med.nyu.edu vilimf01 at mcrcr6.med.nyu.edu
Tue Nov 26 15:05:22 EST 1996


In article <32995F34.85A at Bris.ac.uk>, Andrew Doherty <A.Doherty at Bris.ac.uk> writes:
>>         The other thing that I've just tried is the quick
>> transformation protocol described in the recent Fermentas Newsletter
>> and in NAR 24(3) p536 1996.  Basically, you mix the ligation or DNA
>> with the competent cells, mix, leave on ice 5min, and plate onto
>> prewarmed plates.  This has worked for me the one time I tried it and
>> its a real timesaver.  Be aware that there are cell types that don't
>> like this protocol, and it doesn't work so well for kanamycin
>> resistance (but works fine for amp,tet, cam, DH5alpha, and XL1 blues).
>> 
>>                                 Regards                 SVEN
>> 
>> >
> 
> HI Sven,
> 
> HOw is this a great time saver? The only difference it would make to me
> is that I don't incubate my bugs for an hour or so before plating
> following electroporation. I always use the straight ligation mix to
> transform bugs and it's fine. I must say though, that if it works it
> would save a fortune in electroporation cuvettes!!
> 
> Andy D
> -- 
> *************************************************************
> Dr Andrew Doherty		email -  a.doherty at bris.ac.uk
> Dept. Anatomy			Tel (0117)9287421
> School of Medical Sciences	Fax (0117)9287402
> University of Bristol
> University Walk
> Bristol UK
> BS8 1TD
> *************************************************************

Well, I don't think it will give you efficiencies in the
electroporation range for those types of bugs.  The papers describe
use with chemically competent cells (CaCl2).  I can say that with my
cells (Inoue competent) I got about the same efficiency as with the
regular heat shock (albeit from two different, but similarly processed
ligations).  In any case, I wouldn't throw out the electroporator just
yet.  With regard to the timesaving, an hour and a half vs 5 min - do
the math.

				Regards			SVEN






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